Protein secretion

ABSTRACT

The invention relates to a novel prokaryotic expression system characterized by alterations to the dlt operon whereby expression of genes encoded therein may be regulated to limit the availability of polypeptides encoded by said genes.

The invention relates to a novel prokaryotic expression system andproteins expressed thereby.

The industrial production of proteins has, in many instances, exploitedthe native expression and secretory systems of microorganisms andspecifically bacteria. For example and without limitation the bacteriumBacillus subtilis (B.subtilis) is known to produce and secrete a numberof proteins. One family of these proteins, α-amylases, is of industrialimportance and therefore the harvesting of this secreted protein is anactivity currently undertaken by industry. However, the yield of someα-amylases is significantly reduced by protein degradation during an/orfollowing passage through the cell membrane.

It therefore follows that there is a need to provide a proteinexpression system which enhances the production of native and/orheterologous and/or recombinant protein and more specificallyeffectively enhances the secretion of protein from the cell.

The expression and secretion of heterologous and/or recombinant protein(i.e. proteins that are not native to that particular bacteria)typically involves transformation of a bacterial cell with heterologousDNA with a view to manufacturing or producing heterologous and/orrecombinant proteins.

Microorganisms such as Escherichia coli (bacteria), Saccharomycescerevisiae, Aspergillus nidulans and Neurospora crassa (fungi) have beenused in this fashion. The expression of heterologous protein inprimitive eukaryotes also allows some desirable eukaryoticpost-translational modifications to occur in heterologous and/orrecombinant proteins leading to an increase in the stability of theexpressed proteins and subsequent improvement in yield. More recentlythe use of mammalian and insect cells have been developed to facilitatethe expression of eukaryotic proteins that for various reasons cannot beexpressed in a prokaryotic host cell.

However, the cost effectiveness of producing heterologous and/orrecombinant protein still remains the major advantage offered bygenetically engineered prokaryotic expression systems and indeedsignificant advances have been made in the development of geneticallyengineered E.coli strains that increase the yield of specific proteins.The development of these bacterial strains has also been married with anever increasing development of more efficient vectors adapted tooptimise the expression of recombinant protein. These vectors containpromoter elements that are genetically engineered to create hybridpromoters that can be switched on or off with ease.

However, there are three major disadvantages when using E.coli as ameans of expressing heterologous and/or recombinant protein. Firstly,the high levels of expression lead to a precipitation of recombinantprotein in the bacterial cytoplasm as “inclusion bodies”. This featurewas thought to be advantageous as it can provide a simple means ofseparating the insoluble recombinant protein from the soluble endogenousE.coli protein. However, in reality this advantage is not a generalfeature of the system as in many cases proteins remain an insolubleprecipitate that can only be released into solution by using strongchaotropic agents. This presents a major problem if the protein inquestion is particularly labile and therefore loses biochemical orbiological activity upon denaturation. Secondly, the expression offoreign protein in E.coli leads to rapid degradation of these proteinsvia an efficient proteolytic system. Thirdly, it is known by thoseskilled in the art that E.coli usually does not naturally secreteprotein into its surrounding environment. Therefore, the purification ofnative, heterologous or recombinant protein has the major disadvantagethat the desired protein has to be purified from endogenous E.coliprotein.

E.coli strains have been engineered to allow the expression ofrecombinant proteins that would ordinarily be difficult to express intraditional laboratory strains of E.coli. However, these engineeredE.coli strains are invariably not as biologically disabled astraditional laboratory strains of E.coli and as a consequence requirecontainment levels that are higher than would normally be required.

The identification of alternative prokaryotic host cells and thedevelopment of means that facilitate the production of soluble, intactand biologically active protein is obviously desirable. However, notablythe number of potential prokaryotic host cells is huge.

With a view to producing a novel protein and expression system we havechosen to genetically engineer, as our example, Bacillus, ideallyB.subtilis, in order to provide an expression system that overcomes theproblems of yield associated with prior art systems. We have focussedour attention on providing a Bacillus expression system that producesand ideally secretes protein(s) into the culture medium because thissystem enables an initial purification of the manufactured protein dueto the absence of contaminating endogenous bacterial protein(s) andother macromolecules.

The biochemical composition of the B.subtilis cell wall is quitedifferent from that of E.coli. The cell walls of E.coli and B.subtiliscontain a framework that is composed of peptidoglycan, a complex ofpolysaccharide chains covalently cross-linked by peptide chains. Thisforms a semi-rigid structure that confers physical protection to thecell since the bacteria have a high internal osmotic pressure and can beexposed to variations in external osmolarity. In Gram-positive bacteria,such as the members of the genus Bacillus, the peptidoglycan frameworkmay represent as little as 50% of the cell wall complex and thesebacteria are characterised by having a cell wall that is rich inaccessory polymers such as wall teichoic acids. In addition, teichoicacids may be attached to the outside of the cytoplasmic membrane in theform of lipoteichoic acids or membrane anchored wall teichoic acids.

Teichoic acids are simple polymers of alditol phosphate molecules linkedto each other by phosphodiester bridges. The free hydroxyl groups of thealditol phosphate backbone may be occupied by alanine or sugar residues.The alanylation of teichoic acids has a major effect of neutralising thenegative charge conferred by adjacent phosphate residues, therebyreducing the overall negative charge of the cell wall.

The cell wall therefore provides, amongst other things, protection tothe cell membrane to prevent rupture. The peptidoglycan frameworkrepresents upto approximately 50% of the cell wall mass. The remainingwall material consists of components which differ significantly betweenGram negative (E.coli) and Gram positive (B.subtilis) bacteria.B.subtilis, and many other Gram positive bacteria, is characterised byhaving a cell wall that is rich in the accessory molecule teichoic acid.

The alanylation of teichoic acids is controlled by theD-alanyl-lipoteichoic acid (dlt) operon, a cluster of five genesencoding proteins necessary for the alanylation of teichoic acid. Thegenes are termed dltA, dltB, dltC, dltD and dltE. With the exception ofdltE, each of these genes have known functions, Perego et.al 1995,please see FIG. 1.

The partial or complete deletion of any individual member of the dltoperon, with the exception of the dltE, completely inhibits thealanylation of teichoic acid. However, there is no obvious phenotypiceffect of deleting one or more of the dltA-D genes other than theinhibition of alanylation and consequential changes in the overallsurface charge. Cell division and growth are apparently unaffected inB.subtilis.

An unrelated gene, prsA, encodes a cell membrane located chaperone likemolecule. The protein is involved in the folding of secreted proteins onthe extracytoplasmic side of the cytoplasmic membrane (Kontinen et.al.1991; Jacobs et.al 1993). Sequence homology with severalpeptidyl-prolyl-isomerases suggests that the PrsA protein is involved inthe isomerisation of proline residues between cis and trans isomers insecreted proteins. A number of mutations have been identified and arerelatively easy to determine by the diminished ability of prsA mutantsto secrete α-amylase. An example of one such mutation is prsA3, Kontinenand Sarvas, 1993. Interestingly although mutants possessing a mutationin prsA show a decrease in the secretion of α-amylase and exoprotease,some secreted proteins, for example penicillinase, are unaffected. Thissuggests that PrsA is

selectively involved in the secretion of proteins and that thisselection may be determined by the number/position of proline residuesor nature of its nearest neighbours in secreted proteins.

In an attempt to identify second site suppressors of prsA3 we haverandomly mutagenised B.subtilis with the mini-transposon, Tn-10. Thistransposon randomly integrates into bacterial DNA and, as long as anessential gene is not disrupted, the Tn10 mutants are viable.

We have taken a B.subtilis strain carrying the prsA3 mutation andidentified Tn10 integration mutants that show enhanced secretion ofα-amylase into the culture medium. One such mutant, designated IH7231was further analysed by DNA sequencing of the flanking regions ofrescued Tn10 DNA to identify the site of integration. After sequencecomparisons of the rescued DNA with the published B.subtilis genomicsequence we suprisingly found the rescued sequence to be homologous tothe dltD gene of the dlt operon, Perego et.al. 1995.

The published prior art does not indicate an involvement of the dltoperon in the secretion of proteins from B.subtilis. Indeed the onlyapparent phenotypic change in B.subtilis cells disrupted for any of thedltA-D genes is the failure of the cell to add D-alanine to wall orlipo-teichoic acids. It is therefore both suprising and intriguing thatthe disruption of a dlt gene should have this phenotype.

It is therefore an object of this invention to develop a means ofexpressing recombinant protein in a prokaryotic expression system thatallows the production of proteins and/or polypeptides in a biologicallyactive form and at high concentration.

It is further object of the invention to develop a prokaryoticexpression system that enables the secretion of native, heterologous orrecombinant protein into culture medium to facilitate the purificationof proteins and/or polypeptides that retains biological activity.

According to a first aspect of the invention there is provided abacterial strain whose genome includes the dlt operon wherein the operonhas been altered by substitution and/or deletion and/or insertion and/ormutation so that either production of at least part of at least oneproduct(s)encoded by said dlt operon is prevented or at least part of atleast one gene product is non-functional to the extent that the use ofthe strain to produce native, heterologous or recombinant protein isfacilitated.

Reference hereto the term bacterial strain includes reference to anybacterial strain but ideally a Gram-positive bacterial strain and, moreideally, but not obligatory, a bacterial strain of the genus Bacillus.

It will be apparent to those skilled in the art that where heterologousand/or recombinant protein is to be produced the said bacterial strainwill be transformed so as to include DNA encoding at least one selectedheterologous and/or recombinant protein.

It will also be apparent to one skilled in the art that said alterationmay be to at least one of the dlt A-E genes as represented in FIG. 1. Sothat said alteration ideally leads to a failure of said strain to addD-alanine to teichoic acid.

In a further preferred embodiment of the invention said alteration is toat least part of the dlt A-E genes.

In yet a further preferred embodiment of the invention said alterationis to at least part of dltA and/or dltB and/or dltC and/or dltD and/ordltE, preferaby dltB but ideally dltD.

It will be apparent that means to effect said alteration to the dltoperon are well known in the art. For example, and not by way oflimitation, the insertion of genetic material into the dlt operon may beundertaken by transposon integration. Additionally or alternatively, theoperon may be altered to provide for deletion of at least part of atleast one gene located in the dlt operon by homologous recombinationwith at least one suitably designed vector and/or the replacing of atleast part of at least one gene located in the dlt operon withhomologous DNA carrying, for example, a translation termination codonthus preventing synthesis of a functional protein. Additionally oralternatively, the operon may be altered by base substitution and/ormutation by random or site-directed mutagenesis.

In yet a further preferred embodiment of the invention said dlt operonis altered by way of alteration of an expression control sequence,ideally a promoter, such that the promoter is made responsive to aspecific signal, for example, the operon may be placed under the controlof an inducible promoter such that expression of the operon encoded geneproducts may be selectively controlled.

It is well known in the art that means to place the aforedescribed genesunder the control of regulatable promoters exist and include those meansdescribed for placing the dlt operon under regulatable expression.

The dlt operon is controlled by a single promoter element regulated bythe transcription factor (sigma D or σ^(D)), therefore the aboveembodiment of the invention may comprise replacement of sigma D or σ^(D)with, for example, and not by way. of limitation, an IPTG induciblepromoter. By placing the dlt operon under the control of an IPTGinducible promoter the expression of proteins encoded by the dlt operoncan simply be induced by addition of IPTG.

Alternatively the expression of the dlt operon may be repressed by, forexample and not by way of limitation, incorporation of a tetracyclineresponsive element. The tetracycline responsive element binds the TETrepressor protein to prevent transcription from a promoter containingthe responsive element. Therefore a bacterial strain according to theinvention could be further genetically engineered to contain a geneexpressing the TET repressor and a dlt operon containing the TETresponsive element.

Methods to manipulate bacterial promoters in the aforedescribed mannerare well known in the art.

In an alternative embodiment of the invention said alteration accordingto the invention involves manipulation of the native promoter element ina manner that results in the provision of a non-functional promoterelement incapable of initiating transcription at the dlt operon.

In an alternative embodiment of the invention said alteration of anexpression control sequence is an alteration to at least one mRNAstabilising sequence element located in non-coding regions of the dltoperon. More ideally still, said non-coding regions are located in the5′ or 3′ non-translated regions of mRNA molecules encoded by the dltoperon.

It is well known in the art that the stability of bacterial mRNA iscontrolled to a greater extent by sequences located at the 3′ end ofmRNA which interact with proteins to either stabilise or de-stabilisemRNA molecules. The selective deletion, substitution, insertion ormutation of the sequences may de-stabilise MRNA molecules derived fromthe dlt operon but in any event results in decreasing and/or inhibitionin the expression of dlt encoded proteins.

In yet a further preferred embodiment of the invention said alterationof an expression control sequence is to 5′ translation control sequencesof mRNAs encoded by the dlt operon.

Translation control sequences are well known in the art and include, byexample and not by way of limitation, Shine Dalgarno sequence motifsfound near the translation start codon in many prokaryotic mRNA's.

In a third aspect of the invention there is provided a method forproducing a desired native, heterlogous or recombinant protein and/orpolypeptide, wherein bacteria, as aforedescribed, is used for theproduction of the protein and/or polypeptide by;

i optionally, transforming a bacterial strain according to the inventionwith a suitable vector genetically engineered to facilitate expressionof said polypeptide;

ii culturing said bacterial strain under conditions conducive to theproduction of said polypeptide; and

iii recovering and purifying the said polypeptide from said bacterialstrain and/or growth medium.

The introduction of a vector into a bacterial strain according to theinvention may be by any method known in the art, such as conventionaltransformation methods, electroporation, conjugation or protoplasttransformation. The expression construct may be a plasmid or any othervector suitable for the specific method used for introducing saidexpression construct into a bacterial cell.

In essence the invention provides a bacterial strain, ideally aB.subtilis strain, that has been mutated to provide a bacterial strainthat is facilitated in the secretion of native, heterologous orrecombinant protein into surrounding growth medium.

An embodiment of the invention will now be described by way of exampleonly with reference to the following figures wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the dit operon of B.subtilis;

FIG. 2 represents the accumulation of heterologous and/or recombinantα-amylase (Amy Q) in culture media of a B.subtilis strain disrupted fordltD;

FIG. 3 represents the accumulation heterologous and/or recombinantα-amylase (Amy L) in culture media of a B.subtilis strain disrupted fordltB;

FIG. 4 represents the accumulation of heterologous and/or recombinantpneumolysin in culture media of a B.subtilis strain disrupted for dltD;

FIGS. 5a-5 b. FIG. 5a represents the alanine content of B.subtilis cellwalls of wild type and a strain disrupted for the dltB gene; FIG. 5brepresents the accumulation of heterologous and/or recombinant α-amylase(AmyL) in culture media of wild type B.subtilis and a strain disruptedfor the dltB gene; and

FIGS. 6a and 6 b represents a comparison of the synthesis and release ofheterologous and/or recombinant α-amylase (AmyL) from wild typeB.subtilis and a strain disrupted for the dlt B gene.

MATERIALS AND METHODS

TABLE 1 Bacterial strains and plasmids B. subeilis strainGenotype/Phenotype IH6531 glyB133 hisA1 trpC2, pKTH10 IH7144 glyB133hisA1 prsA3, pKTH10 IH7200 glyB133 hisA1 prsA3, pKTh10, pHV1248 IH7231glyB133 hisA1 prsA3 dltD::miniTn10, PkTh IH7347 glyB133 hisA1 trpC2,pKTH239 IH7378 glyB133 hisA1 trpC2 dltD::miniTn10, pKTH10 IH7379 glyB133hisA1 trpC2 dltD::miniTn10, pKTH239 JH642 TrpC2 pheA1 DLT71 JH642 withprom, dltB DLT72 JH642 with dltB DLT74A JH642 with dltDE DLT76 JH642with dlTE DLT77 JH642 with dltC DN1885 AmyE DN1885 xylR::pKS402 DN1885with chromosomal integration of pKS402 at xylR DN1885 xylR::pKS402DN1885 dltB with chromosomal integration dltB of pKS402 at xylR DN1885xylR::pKS405B DN1885 with chromosomal integration of pKS405B at xylRDN1885 xylR::pKS405B DN1885 dltB with chromosomal integration dltB ofpKS405B at xylR DN1885 xylR::pKS408 DN1885 with chromosomal integrationof pKS408 at xylR DN1885 xylR::pKS408 DN1885 dltB with chromosomalintegration dltB of pKS408 at xylR Plasmids pKS402 Ap^(r), Km^(r),amyLQS50.1, xylR pKS405B Ap^(r), Km^(r), amyLQS50.5, xylR pKS408 Ap^(r),Km^(r), amyL, xylR pKTh10 Km^(r), amyQ pKTh239 Km^(r), P_(amyQ)-pnlpHV1248 Em^(r), Cm^(r) miniTn10, ori-pE194ts

Growth Media

B.subtilis and E.coli were maintained on antibiotic medium number 3(Difco) solidified with 1.5% w/v agar and containing 1% w/v solublestarch. Batch cultures were grown in 2×YT broth buffered with 0.2M MESpH 6.5 which contained; tryptone (1.6% w/v), yeast extract (1.0% w/v)and NaCl (0.5% w/v). Where required antibiotics were included in thegrowth media at the following final concentrations: chloramphenicol6μg/ml, ampicillin 100μg/ml and erythromycin 1μg/ml. Xylose (1% w/v) wasadded to induce the synthesis of α-amylase from a xylose-induciblepromoter.

DNA Manipulations and Bacterial Transformation

Restriction digestion, DNA fragment purification, ligation andtransformation of E.coli were carried out as described previously(Sambrook et al., 1989). Chromosomal DNA was isolated from B.subtilisusing the IGi Genomic extraction kit (Immunogen International) or asdescribed in Marmur, 1961. PCR was carried out with Taq DNA polymerase(Appligene) using B.subtilis DN1885 chromosomal DNA or plasmid pEV1248as the template. Plasmid DNA was purified from E.coli and B.subtlis withthe Tip-100 plasmid extraction kit (Qiagen). Oligonucleotide primers forPCR were synthesized using a Beckman Oligo 1000. B.subtilis was grown tocompetence and transformed with integrative plasmids.

α-Amylase Assay

The quantity of secreted α-amylase was quantified using the Phadebasα-amylase assay kit (Kabi Pharmacia). The cells from culture sampleswere pelleted by microcentrifugation and the α-amylase activity in thesupernatant determined as described by the manufacturer.

Origin of α-Amylase. AmyLQS50.5. AmyLQS50.1. AmyL, AmyQ and Pneumolysin

B.subtilis strains used in this study were transformed with expressionvectors encoding polypeptides which are secreted through the B.subtiliscell wall into the extracellular medium. The construction of AmyLQS50.1and AmyLQS50.5 is described in Dr Keith Stephenson's PhD thesis (1996)entitled “Construction and Use of Chimeric α-Amylase to Study ProteinSecretion in Bacillus subtilis. AmyLQS50.5 is a chimeric α-amylase andthe construction is described in “Secretion of Chimeric α-Amylase fromB.subtilis”; AmyL is derived from B.licheniformis; AmyQ is derived fromB. amyloliquefaciens. Pneumolysin is derived from Streptococcuspneumoniae.

Generation of a B.subtilis Strain Disrupted for dltB

The dltB mutation was generated in DN1885 xylR::pKS405B, DN1885xylR::pKS402 or DN1885 xylR::pKS408 (AmyLQS50.5, AmyLQS50.1 and AmyL,respectively ) by transforming DNA from JH642::pDLT72 (Perego et al 1995J Biol Chem 270: 15598-15606). A fragment of JH642::pDLT72 DNA withinsertionally inactivated dltB integrated into the chromosome via adouble cross-over event and this is selected for on nutrient agar platescontaining erythromycin.

Random Mutagenesis With Mini Tn 10 Transposon

Tn mutagenesis was performed as described in (Petit et al., 1990) withthe strain IH7200. IH7200 is a prsA3 mutant, which harbors the plasmidpKTH10 (Palva, 1982) expressing AmyQ and the plasmid pHV1248 carrying atemperature sensitive replicon and the mini-Tn10 transposon (Petit etal., 1990). Bacteria of the strain IH7200 were grown in 10 ml of L-brothsupplemented with kanamycin (10 μg/ml), erythromycin (1 μg/ml) andchloramphenicol (5 μg/ml) up to mid-exponential phase of growth (50Klett units) at 30° C. and then shifted to 51° C. for 2.5 hours. Sampleswere then plated on L-plates containing 9% soluble starch, kanamycin andchloramphenicol (above concentrations), followed by incubation at 51° C.over night. Tn mutants with increased secretion of AmyQ were detectedfrom a larger halo around colonies. A strain identified in this screenwas IH7231.

Identification of Tn 10 Inactivated Gene in IH7231

The gene inactivated in IH7231 was identified by cloning a fragment ofits chromosomal DNA containing the Tn10 transposon into thebacteriophage vector, Lambda GEM11 (Promega). Cloning in the lambdavector was performed according to the manufactures instructions. Plaquescontaining the transposon were identified by their ability to hybridiseto a digoxigenin-labeled probe of the cat gene of pHV1248. DNA wasisolated from a Lambda clone isolate from such a positively hybridisingplaque and the DNA flanking the TN10 insert was sequenced. Comparison ofthe obtained sequence with the sequence of the whole genome of B.subtilis revealed that the gene interrupted by the transposon was dltD,the fourth gene in the dlt operon (Perego et al., 1995) responsible forthe D-alanylation of lipo- and wall teichoic acids. The product of thedltD gene encodes a protein which probably catalyses the transfer ofD-alanine to the lipo- and wall teichoic acids. Inactivation of the dltDgene by insertion with a derivative of the integrative plasmid pMUTINcontaining part of the dltD gene, or inactivation of the second gene inthe operon, dltB, with integration plasmid pDLT72 (Perego et al 1995),produced the same phenotype as that of IH7231 as determined by the haloassay or assay of α-amylase activity in culture medium. We haveconfirmed that the cell walls isolated from the IH6531 dltD::miniTn10(IH7378), IH7231 and DN1885::pDLT72-derivatives encoding AmyL orAmyLQS50 contain no detectable alanine.

D-Alanine Content of Cell Walls of dltB and dltD Deletion Strains

The alanine content of the cell walls of the dltB and dltD mutants wasdetermined according to IC Hancock and IR Poxton (1988) , BacterialCellSurface Techniques, John Wiley and Sons, Chichester. The methodinvolves preparing cell walls by boiling in buffered SDS (sodium dodecylsulphate, pH6.0), hydrolysing the alanine ester linkages with 0.1Msodium pyrophosphate pH 8.3 and then assaying released alanine with thefollowing solutions A-E mixed in the ratio 40:20:10:5:1

A) 0.1M 0.1M sodiun pyrophosphate pH 8.3

B) 0.2 mg/ml FAD in 0.1M sodiun pyrophosphate pH 8.3

C) Horseradish peroxidase (200 U/mg) at 50 micrograms per milliliter inwater

D) Dianisidine sulphate, 5 mg/ml in water

E) D-amino acid oxidase (15 U/mg) at 5 micrograms per milliliter inwater. The reaction is stopped by the addition of 0.1% SDS andabsorbance measured at 460 nm. The results of these assays are presentedin table 1

Results

We have confirmed the beneficial activity of dlt operon knockouts on theproduction of proteins by measuring the production of 3 commerciallyimportant proteins, namely AmyL, AmyQ and Streptococcal pneumolysin.

Increased α-Amylase Secretion in dltD and dltB Gene Disruptans

In the case of AmyQ, wild type (IH6531) and dltD::miniTn10 (IH7378)strains harbouring pKTH10 were cultured for 72 hours in double-strengthL-broth containing starch and 100 mM bis-tris propane, pH 6.5 at 37° C.in shake flasks. α-Amylase was assayed in the culture supernatant usingthe Phadebas assay kit (Pharmacia). The levels of α-amylase were similarin exponential phase and early stationary phase, however, as stationaryphase was prolonged, increasingly higher amounts of α-amylase wereproduced by the strain with the dltD mutation. After 72 hour of growth,the amount of α-amylase in the dltD mutant was about 50% higher than inthe wild type strain, see FIG. 2.

In a complementary experiment, production of AmyL was monitored inDN1885 xylR::pKS408 with or without the dltB inactivated with pDLT72.The strains were grown at 37° C. with shaking in 2×YT containing 0.2MMES buffer at pH 6.5. α-Amylase activity was again measured in theculture medium and the dltB mutant showed an increased in α-amylaseproduction, of about 40% compared to the wild type strain, FIG. 3. Inthe case of wild type and dltB strains encoding a derivative of AmyL,namely AmyLQS50.1, the amount of amylase produced was approximatelydouble.

A comparison of the alanine content of cell walls of B.subtilis wildtype (DN1885 xylR:: pKS405b) and the dlt B gene deletion DN1885 xylR::pKS405B dlt shows that although alanine content is negligible in themutant strain, cell division is unaffected, please see FIG. 5a. Theproduction of AmyL is approximately 2-fold enhanced in the deletionstrain, see FIG. 5b.

The amount and cellular distribution of AmyL synthesized by wild type B.subtilis and the dlt B deletion strain was compared, see FIGS. 6a and 6b. The mutant strain shows both enhanced total synthesis (cellassociated and released) and release of AmyL into culture medium, FIG.6b.

Increased Pneumolysin Secretion in dlt D Gene Disruptants

In the case of streptococcal pneumolysin, strains IH7347 (wild type) andIH7379 (dltD::Tn10) both harboured pKTH239, a derivative of pUB110encoding the structural gene of pneumolysin fused to the amyQ promoterand signal sequence. Cells were grown for 72 hours in double-strengthL-broth containing starch and 100 mM bis-tris propane, pH 6.5 at 37° C.in shake flasks. Sample of culture medium were removed at differenttimes during growth and, after boiling in 1% SDS, were subjected toSDS-PAGE in 12% gels. After electrophoresis, the samples wereimmunoblotted and polypeptides cross-reacting with pneumolysin antiserumwas detected with an enhanced chemoluminesence system (Amersham), FIG.4. The amount of pneumolysin was about 50% higher in the dltD deletionstrain at all stages of growth.

Discussion

We have used a strain of B. subtilis that is severely impaired for thesecretion of α-amylase to identify an operon, the dlt operon, thatinfluences the secretion of proteins from B. subtilis and we havesubsequently verified the involvement of the dlt operon by placing itunder the control of an IPTG inducible promoter to regulate theproduction of dlt encoded products. The observation that the products ofdlt operon affected the secretion of α-amylase in a negative manner wasunexpected since their role in the alanylation of wall and lipo-teichoicacids was already established.

The role of dlt encoded products in alanylation of teichoic acids wasconfirmed by the biochemical analysis of alanine content in the cellwalls of various B.subtilis strains carrying lesions in the dit operon,please see Table 2. A comparison of the doubling time for wild-type anddlt mutant strains shows that the strains carrying lesions in the dltoperon do not appear to be compromised in growth, at least under thesegrowth conditions. The only apparent phenotype of this class of mutationis a reduction in alanylation of teichoic acids and an increase insecretion of selected heterologous/recombinant protein.

Although secretion of α-amylase is affected in exponential phase, theinfluence of lesions in the dlt operon is most pronounced in stationaryphase. This is significant because the stationary phase is the mostproductive phase in commercial fermentations.

The initial experiments showed that a dltD::Tn10 mutant was able toproduce more of the B. amyloliquefaciens α-amylase, the involvement ofthis gene was confirmed by use of two other types of insertion mutant.We have also shown that mutations in the dltB gene affect the synthesisof another α-amylase, that from B. licheniformis, in a similar manner.In both cases we confirmed that the cell walls lacked the D-alaninesubstituents that were present in the wild type.

Finally, we have also shown that strains with the dltD::Tn10 mutationwere able to produce increased amounts of the pneumolysin fromStreptococcus pneumoniae, another example of a commercially importantenzyme which is not related to α-amylase and is derived from a genusother than Bacillus .

The mechanism by which inactivation of the dlt operon affects secretionis not known. It could influence exoproteins directly, for example byincreasing their rate of folding as they emerge on the trans side of themembrane from the secretory translocase. Alternatively, the lack ofalanylation may reduce the activity of wall or membrane protease,modulate the concentration of metal cofactors or increase the wallporosity of the cell wall.

REFERENCES

Jacobs, M., Anderson, J. B., Kontinen, V. P. and Sarvas, M. (1993)Mol.Microbiology, 8: 957-966.

Kontinen, V. P., Saris, P. and Sarvas, M. (1991) Mol.Microbiology,5:1273-128.3

Kontinen, V. P. and Sarvas, M. (1993) Mol.Microbiology, 8: 727-737.

Marmur, J. (1961) J. Mol. Biol. 3: 208-218.

Pavla, I (1982) Gene. 15: 43-51.

Petit, M. A., Bruand, C., Janniere, L. and Ehrlich, D. S. (1990)J.Bacteriol., 172: 6736-6740.

Perego, M., Glaser, P., Minutello, A., Straunch, M. A., Leopold, K. andFischer, W. (1995) J.Biol.Chem, 270: 15598-15606.

TABLE 2 D-alanine content of wall teichoic acids extracted from variousstrains of B. subdlis D-alanine content 7.5 hours 24 hours dlt lesionStrain growth growth JH642 no data 5.7 none DLT71 no data none detectedprom, dltAB DLT72 no data none detected dltB DLT74A no data nonedetected dltDE DLT76 no data 5.65 dltE DLT77 no data none detected dltCDN1885 xylR::pKS402 13.2 5.89 none DN1885 xylR::pKS402 none detectednone detected dltB dltB DN1885 xylR::pKS405B 8.84 4.58 none DN1885xylR::pKS405B none detected none detected dltB dltB DN1885 xylR::pKS40819.62 11.84 none DN1885 xylR::pKS408 none detected none detected dltBdltB IH 6531 14.83 no data none IH 6531 dltD (IH7378) none detected nodata dltD IH 7144 18.25 no data none IH 7144 dltD (1H7231) none detectedno data dltD

We claim:
 1. A method for producing a native, heterologous orrecombinant secreted polypeptide, wherein said method comprises: i)providing a Gram positive bacterial strain which expresses the native,heterologous or recombinant secreted polypeptide and the genome of whichincludes the dlt operon which has been altered by substitution,deletion, insertion and/or mutation, so that at least part of at leastone gene product of the dlt operon is not expressed or is renderednon-functional; ii) incubating said bacterial strain under conditionsconducive to the production of said polypeptide; and iii) recovering andpurifying said polypeptide from said bacterial strain and/or growthmedium.
 2. A method according to claim 1, wherein said bacterial strainis transformed with an expression vector comprising a gene encoding thepolypeptide.
 3. A method according to claim 1, wherein said bacterialstrain belongs to the genus Bacillus spp.
 4. A method according to claim3, wherein said bacterial strain is selected from the group consistingof B.subtilis, B.licheniformis and B.amyloliquefaciens.
 5. A methodaccording to claim 1, wherein at least part of dlt A gene and/or dlt Bgene and/or dlt C gene and/or dlt D gene and/or dlt E gene is altered.6. A method according to claim 5, wherein at least part of the dlt Dgene is altered.
 7. A method according to claim 5, wherein at least partof the dlt B gene is altered.
 8. A method according to claim 1 whereinsaid dlt operon is altered by way of alteration of an expression controlsequence.
 9. A method according to claim 8 wherein said dlt operon isaltered by way of alteration of a promoter control sequence.
 10. Amethod according to claim 9 wherein said promoter control sequence isaltered by incorporation of an inducible promoter sequence element. 11.A method according to claim 9 wherein said promoter control sequence isaltered by the incorporation of a repressor promoter sequence element.12. A method according to claim 9 wherein said promoter control sequenceis altered so as to provide a non-functional promoter control sequence.13. A method according to claim 8 wherein said alteration of anexpression control sequence is an alteration to at least one mRNAstabilising sequence element located in a non-coding region of the dltoperon.
 14. A method according to claim 13, wherein said non-codingregion is located in the 5′ untranslated region of an mRNA moleculeencoded by the dlt operon.
 15. A method according to claim 14 whereinsaid alteration of an expression control sequence is to the 5′translation control sequence of an mRNA encoded by the dlt operon.
 16. Amethod according to claim 1 wherein the polypeptide is an α amylase. 17.A method according to claim 13, wherein said non-coding region islocated in the 3′ untranslated region of an mRNA molecule encoded by thedlt operon.
 18. A method according to claim 13, wherein alterations areeffected in non-coding regions located in the 5′ and 3′ untranslatedregions of an mRNA molecule encoded by the dlt operon.